Immunologic tolerance was conceived as an "indifference or non-reactivity" measured by the acceptance of allogeneic skin grafts. While thought to be due to the lack of immune responsiveness, we now know that, in addition to clonal anergy, active suppression is essential for graft persistence. This proposal focuses on the active, effector mechanisms that underlie the longevity of allografts in the tolerant recipient. The cellular complexity of graft tolerance can be reduced by using well-defined, alloantigen-specific, TCR Tg populations of effector T cells (Teff) and regulatory T cells (Treg). We present a TCR Tg system can be used to study the cellular dynamics of graft rejection and tolerance in vivo, and can help to define the underlying molecular mechanisms of how Treg control Teff function in vivo. The specific aims of this proposal are: 1) To understand how Treg control the initial expansion and effector function of alloreactive CD4+ and CD8+ T cells in the naive and primed host to control graft rejection. Natural Treg (Treg) and adaptive Treg (Tr1) to a single, defined class ll-restricted, allogeneic epitope arrest Teff responses in vitro and in vivo. How Treg to a single defined epitope can arrest allogeneic responses across complete H-2 disparities will be the focus of this aim. The impact of antigen-specific Treg on CD4+ and CD8+ Teff growth, differentiation, effector function and gene expression will be explored, as well as the conditions that control Treg themselves. Finally, data suggests that Treg may also have the capacity to control memory T cell responses and thus it will be determined if Treg can mitigate graft rejection in the sensitized host. 2) To delineate the cellular mechanisms that allow for long-lived graft survival. While Treg initially establish a state of tolerance, evidence suggest that Treg are catalytic and "infectious". Studies are proposed to delineate how suppression spreads" over time. By creating alloreactive TCR Tg T cells (Treg and Teff) containing the Foxp3gfp knock- in allele (that expresses GFP upon Foxp3 expression), the differentiation of Teff to antigen-specific adaptive Treg in vivo can be determined. The functional importance of distinct Treg subsets to graft survival will be determined by using genetic strategies to delete defined Treg subsets following transfer. We hypothesize that over time, the relevant Treg subsets to graft survival will change. 3. To define the contact-dependent and soluble factors that control alloreactive T cell responses and allow long-lived graft survival. We focus on known (TGFb and IL-10) and new mediators with the intent of solving how these molecules control tolerance. Incisive studies to determine the contribution of TGFb and IL-10 to "infectious" aspects of tolerance are described. Second, data shows that granzyme B (GZB) production by Treg is involved in graft tolerance in vivo. Studies are presented to identify the cellular targets of GZB and its mode of action in sustaining graft survival. New evidence strongly implicates IL-9 and mast cells as elements that may be important to peripheral tolerance, and studies are presented to functionally implicate their involvement.